Sialidase, also called neuraminidase (NA), is an exo-glycosidase that catalyzes the hydrolysis of terminal sialic acid residues from the oligosaccharides of glycoconjugates. Sialidases are widely expressed for various functions. (R. K. Y. M. Saito, Biochemistry and function of sialidases, Plenum Press: New York, 1995.) Many pathogens, such as viruses, bacteria, and protozoa, produce sialidases for invasion, nutrition, detachment, and immunological escape. (E. Severi, et al. Microbiology 2007, 153, 2817)
Mammalian sialidases also have been implicated in many biological processes, including regulation of cell proliferation/differentiation, modulation of cell adhesion, metabolism, and immunological functions. (T. Angata and A. Varki, Chem. Rev. 2002, 102, 439. A. Varki, Nature 2007, 446, 1023.) Four types of sialidases have been identified and characterized in mammalians. These sialidases are encoded by different genes and expressed at different intracellular locations as lysosomal (Neu1), cytosolic (Neu2), plasma-membrane (Neu3), and mitochondrial/lysosomal (Neu4) enzymes. Although these enzymes share a common mechanism of actions, they have little overlapped functions, probably due to differences in subcellular distribution, pH optimum, kinetic properties, and substrate specificities. (T. Miyagi and K. Yamaguchi, Glycobiology 2012, 22, 880.) The regulation and detailed functions of these enzymes are largely undefined. (E. Monti, et al. Adv. Carbohydr. Chem. Biochem. 2010, 64, 403.)
Alterations in sialidase activities have been implicated in different diseases. For example, elevated sialidase activities have been reported in BHK-transformed cells and in human breast/colon cancer tissues. (C. L. Schengrund, et al. J. Biol. Chem. 1972, 247, 2742. H. B. Bosmann and T. C. Hall, Proc. Natl. Acad. Sci. USA 1974, 71, 1833.) Animal studies also suggest the roles of sialidases in tumorigenic transformation and tumor invasion. Biochemical characterizations of mammalian sialidases suggest that increases in Neu3 are involved in colon, renal, and prostate cancers. Transfection of the Neu3 gene into cancer cells leads to protection against apoptosis by increased Bcl-2 expression and decreased activity of caspase-3/-9. (T. Miyagi, Proc. Jpn. Acad. Ser. B Phys Biol. Sci. 2008, 84, 407.)
Furthermore, Neu3 overexpression increases cell motility and invasion by modulation of EGF receptor phosphorylation and Ras activation. (T. Wada, et al. Oncogene 2007, 26, 2483. T. Miyagi, et al. J. Biochem. 2008, 144, 279.) In contrast to the apparent Neu3 promotion in cancer progressions, other sialidases play roles in cancer reduction through accelerated cell apoptosis, differentiation, and suppression of cell invasion. (T. Miyagi, et al. Glycoconj. J. 2004, 20, 189.)
In other aspects, deficiency of the lysosomal sialidase (Neu1) is considered as a major cause for sialidosis, which is an inherited lysosomal storage disease resulting in excessive accumulation of sialylglycoconjugates and development of progressive neurosomatic manifestations. (G. H. Thomas, Disorders of Glycoprotein Degradation: α-Mannosidosis, β-Mannosidosis, Fucosidosis, and Sialidosis, 8 ed., McGraw-Hill: New York, 2001.)
Activity-based protein profiling (ABPP) is a functional proteomic technology that uses chemical probes for specific enzymes. (M. J. Evans and B. F. Cravatt, Chem. Rev. 2006, 106, 3279.) An ABPP probe is typically composed of two elements: a reactive group and a tag. The reactive group is designed based on the catalytic mechanism of the target enzyme, and it usually contains an electrophile that can covalently link to nucleophilic residues in the enzyme active site. The tag may be either a reporter such as a fluorophore or an affinity label such as biotin. The tag can incorporate an alkyne or azide moiety for subsequent modification by the Cu(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) to introduce a reporter. (V. V. Rostovtsev, et al. Angew. Chem. Int. Ed. 2002, 41, 2596. H. C. Kolb and K. B. Sharpless, Drug Discov Today 2003, 8, 1128.)
ABPP probes can be useful tools to monitor specific enzyme changes in association with certain biological states, such as cancerous status, and responses to stimulants. ABPP probes have been developed for many enzyme classes, including serine hydrolases (Y. Liu, et al. Proc. Natl. Acad. Sci. USA 1999, 96, 14694. D. Kidd, et al. Biochemistry 2001, 40, 4005), cysteine proteases (D. Greenbaum, et al. Mol. Cell. Proteomics 2002, 1, 60), protein phosphatases (C. Walls, et al. Methods Mol. Biol. 2009, 519, 417. K. A. Kalesh, et al. Chem. Commun. 2010, 46, 589), oxidoreductases (G. C. Adam, et al. Nat. Biotechnol. 2002, 20, 805), histone deacetylases (C. M. Salisbury and B. F. Cravatt, Proc. Natl. Acad. Sci. USA 2007, 104, 1171), kinases (M. P. Patricelli, et al. Biochemistry 2007, 46, 350), metalloproteases (S. A. Sieber, et al. Nat. Chem. Biol. 2006, 2, 274), and glycosidases. (C. S. Tsai, et al. Org. Lett. 2002, 4, 3607. D. J. Vocadlo and C. R. Bertozzi, Angew. Chem. Int. Ed. 2004, 43, 5338. K. A. Stubbs, et al. J. Am. Chem. Soc. 2008, 130, 327. M. D. Witte, et al. Nat. Chem. Biol. 2010, 6, 907.)
Two types of sialidase ABPP probes, the quinone methide and the photoaffinity labeling probes have been reported. (G. T. van der Horst, et al. J. Biol. Chem. 1990, 265, 10801. C. P. Lu, et al. Angew. Chem. Int. Ed. 2005, 44, 6888. R. Kannappan, et al. Biol. Pharm. Bull. 2008, 31, 352.) These probes often have problems in non-specific labeling when used in complex protein samples, such as cell lysates. In addition, these probes cannot be applied to in situ labeling experiments because they are impermeable to cell membranes.
For sialidase profiling under physiological conditions, target-specific and cell-permeable ABPP probes are needed to study sialidase changes in living cells. Recently, Withers and coworkers have used 3-fluorosialyl fluoride as an effective inhibitor against Trypanosoma cruzi trans-sialidase (TcTs). (A. G. Watts, et al. J. Am. Chem. Soc. 2003, 125, 7532. S. Buchini, et al. Angew. Chem. Int. Ed. 2008, 47, 2700.)